Crop–Weed Introgression Plays Critical Roles in Genetic Differentiation and Diversity of Weedy Rice: A Case Study of Human-Influenced Weed Evolution

Simple Summary To generate knowledge on how human activities influence plant evolution in agroecosystems, we analyzed allelic introgression from japonica rice varieties into the indica type of weedy rice, and the impact of crop-to-weed introgression on the genetic differentiation and diversity of the weedy populations in Jiangsu Province of China, based on InDel (insertion/deletion) and SSR (simple sequence repeat) molecular fingerprints. Results from these analyses indicated a positive correlation between increased introgression from japonica rice varieties and genetic differentiation in weedy rice. In addition, increased crop-to-weed introgression formed a parabola pattern of dynamic genetic diversity in weedy rice. Our case study indicated that human activities such as the frequent change in crop varieties can influence the evolution of their conspecific weeds through crop-to-weed introgression, which promotes their genetic differentiation and dynamics of genetic diversity in agroecosystems. Abstract As an important driving force, introgression plays an essential role in shaping the evolution of plant species. However, knowledge concerning how introgression affects plant evolution in agroecosystems with strong human influences is still limited. To generate such knowledge, we used InDel (insertion/deletion) molecular fingerprints to determine the level of introgression from japonica rice cultivars into the indica type of weedy rice. We also analyzed the impact of crop-to-weed introgression on the genetic differentiation and diversity of weedy rice, using InDel (insertion/deletion) and SSR (simple sequence repeat) molecular fingerprints. Results based on the STRUCTURE analysis indicated an evident admixture of some weedy rice samples with indica and japonica components, suggesting different levels of introgression from japonica rice cultivars to the indica type of weedy rice. The principal coordinate analyses indicated indica–japonica genetic differentiation among weedy rice samples, which was positively correlated with the introgression of japonica-specific alleles from the rice cultivars. In addition, increased crop-to-weed introgression formed a parabola pattern of dynamic genetic diversity in weedy rice. Our findings based on this case study provide evidence that human activities, such as the frequent change in crop varieties, can strongly influence weed evolution by altering genetic differentiation and genetic diversity through crop–weed introgression in agroecosystems.

Jiangsu Province (JS) is an important rice planting and production region in China, where indica rice varieties were prominently cultivated traditionally [33][34][35][36][37]. The gradual replacement of rice seedling transplanting by direct seeding in JS for the past decades largely promoted the emergence and infestation of weedy rice in rice fields, even though farmers have used commercial and certified rice seeds, and applied herbicides to control weeds [29,33,34]. Historically, no natural distribution of wild Oryza species has been reported in this province. Studies have indicated that JS weedy rice has its dedomestication origins, meaning that JS weedy rice has evolved from the domesticated rice varieties [38][39][40][41], such as the origins of weedy rice from other rice planting regions (e.g., northeastern China) where no wild Oryza species are distributed either [21,29,31]. Given that no wild rice species are naturally distributed in these regions, weedy rice there is genetically similar to the cultivated rice, rather than wild Oryza species (e.g., O. rufipogon and O. nivara) [34,[38][39][40][41][42]. Similar to the characteristics of their co-occurring rice varieties, weedy rice populations found in JS were essentially the indica types [23,34]. Since the end of the 1950s, japonica rice varieties were gradually cultivated in this province due to the favorable taste and high yielding of japonica rice varieties [35,36]. Now, japonica rice varieties are prominently cultivated in JS [33,37]. As a consequence, some weedy rice individuals with japonica characteristics are identified in the rice planting regions of this province [23,34,42]. Obviously, the introgression of japonica-specific alleles from japonica rice varieties into weedy rice with the indica genetic background has played an important role in changing indica-japonica characteristics of weedy rice in JS where the rapid change from planting indica to japonica rice varieties has taken place. Apart from observing the japonica type of weedy rice in JS, our knowledge concerning japonica-crop-to-indica-weed introgression in the evolution of weedy rice is still limited. Therefore, investigating the cultivation history of indica to japonica rice varieties associated with introgression, genetic differentiation, and diversity of weedy rice may provide a deep insight into the role of crop-to-weed introgression in plant evolution.
In this case study, we used the InDel (insertion/deletion) molecular markers [43,44] to determine the genetic differentiation of weedy rice associated with the extent of cropto-weed introgression in JS. We also applied microsatellite (simple sequence repeats, SSR) molecular markers to examine the genetic diversity pattern of weedy rice associated with the extent of crop-to-weed introgression. The primary objectives of the study were to (1) determine the historical changes in rice cultivation associated with indica to japonica varieties in JS; (2) examine whether introgression of japonica alleles from cultivated rice affects the genetic structure of weedy rice; (3) and assess the influences of crop-to-weed introgression on genetic differentiation of weedy rice and on the patterns of genetic diversity in weedy rice. The generated knowledge will facilitate our understanding of the impact of human-influenced evolution on weedy species in agroecosystems.

Historical Changes in Rice Cultivation from Indica to Japonica Varieties in Jiangsu Province
The change in planting areas from indica to japonica rice varieties in Jiangsu Province (JS) was estimated mainly based on the published statistical data, including Jiangsu Agricultural Statistics , Economic Data of Rural Jiangsu Province (1988)(1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998), and Statistical Yearbook of Rural Jiangsu Province (2000-2017) [45][46][47]. In addition, the published books, including The Science of Rice Cultivation in Jiangsu Province, The History of Agricultural Development in Jiangsu Province, and The Documents of Jiangsu Provincial Annals (Agricultural) [48][49][50], were also used to estimate the historical change in rice varieties cultivated in JS. To demonstrate the pattern of the spatial-temporal changes in japonica rice cultivation in JS, we calculated the average areas (by 1000 hectares, kha) of japonica rice cultivation for every 10 years, based on each county as a unit from the 1950s to 2020s. The obtained average data of japonica rice cultivation areas were visually presented in GIS maps with the different units in JS, using the software ArcGIS ver.10.2 [51].

Collection of Plant Materials
Mature seed samples were collected from a total of 36 natural weedy rice populations during October 2020~2021 across the rice planting regions in JS ( Figure 1 and Table S1). The density of weedy rice occurring in the JS rice fields was about 0.5-1.0 plant per 100 m 2 . For sample collection, we included 30~32 randomly selected individuals (samples) from each weedy rice population with the spatial distances >10 m in a sampling rice field (about 6000 m 2 ). In comparison, mature seed samples of the accompanying rice varieties in the same fields were also collected. The spatial distances between the collecting sites (fields) for each weedy rice population were >10 km. In addition, mature seed samples from three weedy rice populations each in Guangdong Province (GD) and northeast China (NEC) were collected to represent the indica and japonica types of weedy rice, respectively (Table S1). Furthermore, mature seeds of 13 typical indica and 13 typical japonica rice varieties from various sources identified by "InDel molecular index" [43] were also included as the references for further analyses (Table S1). changes in japonica rice cultivation in JS, we calculated the average areas (by 1000 hecta kha) of japonica rice cultivation for every 10 years, based on each county as a unit from 1950s to 2020s. The obtained average data of japonica rice cultivation areas were visu presented in GIS maps with the different units in JS, using the software ArcGIS ver.1 [51].

Collection of Plant Materials
Mature seed samples were collected from a total of 36 natural weedy rice populati during October 2020~2021 across the rice planting regions in JS ( Figure 1 and Table  The density of weedy rice occurring in the JS rice fields was about 0.5-1.0 plant per m 2 . For sample collection, we included 30~32 randomly selected individuals (samp from each weedy rice population with the spatial distances >10 m in a sampling rice fi (about 6000 m 2 ). In comparison, mature seed samples of the accompanying rice varie in the same fields were also collected. The spatial distances between the collecting s (fields) for each weedy rice population were >10 km. In addition, mature seed samp from three weedy rice populations each in Guangdong Province (GD) and northe China (NEC) were collected to represent the indica and japonica types of weedy rice, spectively (Table S1). Furthermore, mature seeds of 13 typical indica and 13 typical japon rice varieties from various sources identified by "InDel molecular index" [43] were a included as the references for further analyses (Table S1).  Table S1 (Population ID in the 4th umn).

DNA Extraction, Amplification, and Genotyping
Seeds of weedy rice and cultivated rice were germinated in an illuminated incuba (Percival Scientific, Perry, IA, USA) (25 ± 3 °C) with alternating light/dark (16/8 h). T total genomic DNA was extracted from the 14-day-old fresh seedlings following a mo fied CTAB protocol [52].
Thirty-eight indica-japonica specific InDel primer pairs (Table S2) [43,44], distribu on both arms of each of the 12 rice chromosomes, were selected to determine indica a  Table S1 (Population ID in the 4th column).

DNA Extraction, Amplification, and Genotyping
Seeds of weedy rice and cultivated rice were germinated in an illuminated incubator (Percival Scientific, Perry, IA, USA) (25 ± 3 • C) with alternating light/dark (16/8 h). The total genomic DNA was extracted from the 14-day-old fresh seedlings following a modified CTAB protocol [52].
Thirty-eight indica-japonica specific InDel primer pairs (Table S2) [43,44], distributed on both arms of each of the 12 rice chromosomes, were selected to determine indica and japonica characteristics of all the weedy and cultivated rice samples. In addition, 47 SSR primer pairs (Table S2) from the rice genome distributed across the 12 chromosomes were selected from the Gramene Markers Database (https://archive.gramene.org/markers/, accessed on 20 April 2023) [53] to analyze genetic diversity of weedy rice populations. All the forward primers of the selected InDel and SSR primer pairs were labeled with one of the following fluorescent dyes: FAM (blue), HEX (green), ROX (red), and TAMRA (black), respectively.
Polymerase chain reactions (PCR) were performed in a total volume of 20 µL containing 1 × PCR buffer (with Mg 2+ ), 200 µM dNTPs, 4 µM of each primer, 0.5 U Taq polymerase, 40 ng template DNA, and ddH 2 O to the final volume. Reaction conditions comprised an initial denaturation step for 4 min at 94 • C, followed by 30 cycles of 30 s at 94 • C, 30 s at 52~58 • C, and 30 s at 72 • C, and a final extension step at 72 • C for 10 min. According to the size of the PCR products, labeled PCR products of 3~5 InDel or SSR primer pairs were mixed together in a ratio of FAM:HEX:ROX:TAMRA = 1:3:1:3, then were electrophoresis separated on ABI 3730xl Analyzer (Applied Biosystems, Waltham, MA, USA). For genotyping, the separated InDel or SSR fragments of each sample were scored using the software GeneMapper version 4.1 (Applied Biosystems, Waltham, MA, USA). The frequency of japonica-specific alleles (F j ) of each weedy and cultivated rice sample was calculated based on the "InDel molecular index" [43]. For each InDel locus, the typical indica rice variety (93-11) and the japonica rice variety (Nipponbare) were used as the reference to determine the homozygote indica (II), japonica (JJ), and heterozygote (IJ) genotypes, respectively (for details, see Lu et al. [43]). In this study, the indica-japonica characterization of all weedy and cultivated rice samples was determined as the indica type (F j < 0.25), intermediate type (0.25 ≤ F j < 0.75), and japonica type (F j ≥ 0.75), respectively, based on the average values of F j or "InDel molecular index" of the 38 InDel loci [43,44].

Estimate of Crop-to-Weed Introgression Using the Frequency of Japonica-Specific
Introgression of the japonica-specific alleles was examined based on the InDel data matrix, including JS weedy rice that originally had an indica genetic background and the reference rice samples (26 typical indica and japonica verities), using the software STRUCTURE v.2.3.4 [54]. The STRUCTURE analysis was conducted with the admixture model and the correlated allele frequency model among groups, with an initial burn-in run of 100,000 steps followed by 200,000 MCMC iterations. The group number (K) was set from 2 to 8 with 10 runs for each K value. The appropriate K value was determined by calculating the value of ∆K described in Evanno et al. [55] using the Structure Harvester (https://taylor0.biology.ucla.edu/structureHarvester/ accessed on 20 April 2023).
To estimate the possible application of the frequency of japonica-specific alleles (F j ) for measuring the levels of crop-to-weed introgression from japonica rice varieties, we analyzed the correlation between the F j values [43] and the proportion of introgressed japonica genetic components (alleles) derived from the "inferred ancestry of individuals" cluster data matrix generated in the STRUCTURE analysis [54]. Results of the correlation between the F j values and the proportion of introgressed japonica genetic components (alleles) were visualized by a linear model of Pearson's correlation method using Prism v.8.0.2 [56].

Correlation between Genetic Differentiation and Crop-to-Weed Introgression in JS Weedy Rice
To determine the indica-japonica genetic differentiation pattern of JS weedy rice, we performed a principal coordinate analysis (PCoA) of the genotypic data matrix based on the 38 InDel molecular fingerprints, using the software GenAlEx v.6.5 [57]. The data matrix included weedy rice samples from JS, GD, and NEC, in addition to the typical indica and japonica rice varieties as references. Results from the first two principal coordinates of all studied samples were graphed in a 2-dimensional scatterplot to illustrate the genetic differentiation of JS weedy rice samples.
To estimate correlations between genetic differentiation and crop-to-weed allelic introgression of weedy rice, we established 30 ideally sampled weedy rice populations (ISWPs), Biology 2023, 12, 744 6 of 19 each containing 16 samples randomly drawn from the JS weedy rice pool with a total of 1116 samples. These populations represented the low (F j < 0.25), middle (0.25 ≤ F j < 0.75), and high (F j ≥ 0.75) levels of introgression, respectively. Consequently, there were 10 lowintrogression, 10 middle-introgression, and 10 high-introgression ISWPs established for further correlation analyses. In addition, we randomly selected eight naturally sampled weedy rice populations (NSWPs) from the field sampled JS weedy rice populations based on their F j values, ranging from 0.05 (lowest) to 0.34 (highest), to analyze the relationships between genetic differentiation of weedy rice and crop-to-weed allelic introgression revealed by ISWPs.
The pairwise Wright's F st values [58] were calculated to represent the genetic differentiation (F st ) between ISWPs based on the genotyping data matrixes of both InDel and SSR molecular fingerprints, respectively, using the software GenAlEx v.6.5 [57]. Moreover, the pairwise differences in the F j values were calculated to represent the differences in introgression (F j-d ) between ISWPs based on the same data matrixes. The correlation between genetic differentiation and crop-to-weed allelic introgression was calculated based on the obtained F st and F j-d values of the 30 ISWPs and eight NSWPs, respectively. The obtained correlation was visualized using Pearson's correlation method (linear model) packaged in the software Prism v.8.0.2 [56].

Correlation between Genetic Diversity and Crop-to-Weed Introgression in JS Weedy Rice
To estimate the correlation of genetic diversity (represented by Nei's expected heterozygosity, H e and Shannon's information index, I) [59,60] with the level of crop-to-weed introgression (F j ), we calculated the two genetic diversity parameters using the same ISWPs and NSWPs (see the Section 2.4.3), respectively, based on the genotyping data matrices of both InDel and SSR molecular fingerprints. The association of the genetic diversity parameters (H e and I) with F j values was obtained using the quadratic equation fitting regression analysis for the ISWPs. The obtained results were visualized using a nonlinear model packaged in the software Prism v.8.0.2 [56]. In addition, we also analyzed the association of the genetic diversity parameters (H e and I) with the F j values in the eight NSWPs using Pearson's correlation method (linear model) packaged in the software Prism v.8.0.2 [56] to confirm the correlation pattern obtained based on ISWPs.

Rapid Alteration of Rice Cultivation from Indica to Japonica Varieties in Jiangsu Province
The historical archives indicated that the tradition of rice cultivation in Jiangsu Province (JS) was essentially indica varieties until the early 1950s, when japonica rice varieties were introduced to this province due to their favorable commercial quality (tastes) and high yield. Statistical data between the 1950s and 2010s evidently demonstrated a relatively rapid change in rice cultivation from indica to japonica rice varieties in this province (Table S3 and Figure 2). Starting from the end of the 1950s, the cultivation areas for japonica rice varieties gradually expanded into the areas where the indica varieties were originally cultivated in this province within only a few decades. Consequently, indica rice varieties were almost completely replaced by japonica rice varieties by the end of the 1990s, particularly in some rice cultivation areas, such as the northeast, east, and south part of this province ( Figure 2).
The statistical data also indicated that the alteration of cultivation from indica to japonica rice varieties started in the southern part of JS. Then, the cultivation areas of japonica rice varieties were gradually extended northward to cover the rice cultivation areas in the whole province. The cultivation areas for japonica rice varieties increased from only 3.4 kha (1000 hectares) in the 1950s to 2015.3 kha in the 2010s. In contrast, the cultivation areas for indica rice varieties dramatically decreased from 1988.5 kha in the 1950s to 290.0 kha in the 2010s (Table S3). Therefore, the different durations of rice cultivation for japonica varieties in different areas could cause the different levels of japonica-specific allelic introgression into the indica type of weedy rice cooccurring with the japonica rice varieties in these areas.
Biology 2023, 12, x FOR PEER REVIEW 7 of 1 cultivated in this province within only a few decades. Consequently, indica rice varietie were almost completely replaced by japonica rice varieties by the end of the 1990s, partic ularly in some rice cultivation areas, such as the northeast, east, and south part of thi province ( Figure 2). The statistical data also indicated that the alteration of cultivation from indica to ja ponica rice varieties started in the southern part of JS. Then, the cultivation areas of japonic rice varieties were gradually extended northward to cover the rice cultivation areas in th whole province. The cultivation areas for japonica rice varieties increased from only 3. kha (1000 hectares) in the 1950s to 2015.3 kha in the 2010s. In contrast, the cultivation area for indica rice varieties dramatically decreased from 1988.5 kha in the 1950s to 290.0 kh in the 2010s (Table S3). Therefore, the different durations of rice cultivation for japonic varieties in different areas could cause the different levels of japonica-specific allelic intro gression into the indica type of weedy rice cooccurring with the japonica rice varieties i these areas.

Patterns of Introgression from Japonica Rice Varieties to Weedy Rice
In general, results based on the calculated "InDel molecular index" clearly indicate that each of the examined cultivated rice and weedy rice samples from Guangdong Prov ince (GD), northeast China (NEC), and JS had their distinct indica, japonica, or indica-japon ica (determined as intermediate) characteristics (Table S1). This characterization was base on the "InDel molecular index" or examined the frequency of japonica-specific alleles (F at the 38 InDel (insertion/deletion) loci across the rice genome. The Fj values of the typica indica rice varieties and weedy rice samples from GD were equal to 0 or close to zero whereas the Fj values of the typical japonica rice varieties and weedy rice samples from NEC were equal to one or close to one (Table S1). These results suggest that the referenc cultivated rice and weedy rice samples from GD or NEC rice cultivation regions had iden tical and unique indica or japonica characteristics, respectively, which set up an ideal stand ard for a comparison of the indica-japonica characteristics of JS weedy rice samples. How ever, the Fj values of the 1116 weedy rice samples from JS ranged from 0 to 0.97 (Table S1 although, most of the samples still showed their indica characteristics (Table S1). In the J

Patterns of Introgression from Japonica Rice Varieties to Weedy Rice
In general, results based on the calculated "InDel molecular index" clearly indicated that each of the examined cultivated rice and weedy rice samples from Guangdong Province (GD), northeast China (NEC), and JS had their distinct indica, japonica, or indica-japonica (determined as intermediate) characteristics (Table S1). This characterization was based on the "InDel molecular index" or examined the frequency of japonica-specific alleles (F j ) at the 38 InDel (insertion/deletion) loci across the rice genome. The F j values of the typical indica rice varieties and weedy rice samples from GD were equal to 0 or close to zero, whereas the F j values of the typical japonica rice varieties and weedy rice samples from NEC were equal to one or close to one (Table S1). These results suggest that the reference cultivated rice and weedy rice samples from GD or NEC rice cultivation regions had identical and unique indica or japonica characteristics, respectively, which set up an ideal standard for a comparison of the indica-japonica characteristics of JS weedy rice samples. However, the F j values of the 1116 weedy rice samples from JS ranged from 0 to 0.97 (Table S1); although, most of the samples still showed their indica characteristics (Table S1). In the JS weedy rice pool, 91% were the indica type (F j < 0.25),~7% were the intermediate type (0.25 ≤ F j < 0.75), and 2% were the japonica type (F j ≥ 0.75) ( Table 1). In addition, most (~81%) cooccurring rice cultivars from JS belonged to the japonica type (Table S1). These results clearly indicated the introgression of japonica-specific alleles from japonica rice varieties into the original indica type of weedy rice populations in the JS rice cultivation region.
To determine the level of introgression of japonica-specific alleles into weedy rice in JS rice fields, where weedy rice was originally composed of the indica genetic background, we conducted the STRUCTURE analysis using the admixture model based on the data matrices of the InDel molecular fingerprints. Results from the STRUCTURE analysis demonstrated the distinct genetic components of the typical indica (red and blue) and japonica (green) rice varieties (references) at the most optimal K value (K = 3) and their neighboring K values (K = 2, K = 4). The findings suggested the distinct genetic components and substantially diverged genetic relationships of the indica and japonica cultivated rice samples used as references (Figure 3). The distinguishable indica and japonica genetic components set up an excellent standard for studying the introgression of japonica-specific alleles (green) into the indica genetic background (red and blue) of weedy rice samples/populations. To determine the level of introgression of japonica-specific alleles into weedy rice in JS rice fields, where weedy rice was originally composed of the indica genetic background, we conducted the STRUCTURE analysis using the admixture model based on the data matrices of the InDel molecular fingerprints. Results from the STRUCTURE analysis demonstrated the distinct genetic components of the typical indica (red and blue) and japonica (green) rice varieties (references) at the most optimal K value (K = 3) and their neighboring K values (K = 2, K = 4). The findings suggested the distinct genetic components and substantially diverged genetic relationships of the indica and japonica cultivated rice samples used as references (Figure 3). The distinguishable indica and japonica genetic components set up an excellent standard for studying the introgression of japonica-specific alleles (green) into the indica genetic background (red and blue) of weedy rice samples/populations.  As expected, weedy rice samples collected from different locations in JS showed their distinct genetic components (Figure 3). Compared with the genetic components of the references (indica: red and blue, japonica: green), the weedy rice samples from JS showed both indica and japonica genetic components ( Figure 3). Apparently, most JS weedy rice samples had indica genetic components, suggesting their close genetic affinity with their originally cultivated indica rice varieties ( Figure 3). However, some JS weedy rice samples exhibited an admixture of indica-japonica genetic components, indicating the different degree of introgression from japonica rice varieties to the indica type of weedy rice. In addition, the admixture genetic components of the japonica weedy rice also ruled out the possible contamination of japonica weedy rice seeds that should not have admixture components. Noticeably, the typical japonica rice reference varieties showed a distinctly unique genetic component (green) with nearly no admixture; although, the typical indica rice reference varieties showed somehow other genetic components with an extremely low level of admixture at the K = 3 and K = 4 ( Figure 3). These results provide opportunities to analyze the level of introgression of the japonica-specific alleles in the JS weedy rice samples, most of which had indica genetic components.
To determine whether the frequency of the japonica-specific alleles (F j ) obtained based on the "InDel molecular index" could be directly used for estimating the level of allelic introgression from japonica varieties into the indica type of weedy rice, we analyzed the correlation between the obtained F j values and the ratios of the introgressed japonicacomponent of weedy rice samples extracted from the "ancestry of individuals" cluster data matrix in the STRUCTURE analysis. Results from the correlation analysis indicated a significant positive correlation (R 2 = 0.94, p < 0.001) between the F j values and the ratios of introgressed japonica components (Figure 4). This finding suggests that the F j values calculated based on the "InDel molecular index" (Table S1) could be used to estimate the level of japonica-specific allelic introgression for further analyses, particularly the relationships of crop-to-weed introgression with genetic differentiation and genetic diversity in JS weedy rice based on the F j values.
an admixture of indica-japonica genetic components, indicating the different degree of in-trogression from japonica rice varieties to the indica type of weedy rice. In addition, the admixture genetic components of the japonica weedy rice also ruled out the possible contamination of japonica weedy rice seeds that should not have admixture components. Noticeably, the typical japonica rice reference varieties showed a distinctly unique genetic component (green) with nearly no admixture; although, the typical indica rice reference varieties showed somehow other genetic components with an extremely low level of admixture at the K = 3 and K = 4 ( Figure 3). These results provide opportunities to analyze the level of introgression of the japonica-specific alleles in the JS weedy rice samples, most of which had indica genetic components.
To determine whether the frequency of the japonica-specific alleles (Fj) obtained based on the "InDel molecular index" could be directly used for estimating the level of allelic introgression from japonica varieties into the indica type of weedy rice, we analyzed the correlation between the obtained Fj values and the ratios of the introgressed japonica-component of weedy rice samples extracted from the "ancestry of individuals" cluster data matrix in the STRUCTURE analysis. Results from the correlation analysis indicated a significant positive correlation (R 2 = 0.94, p < 0.001) between the Fj values and the ratios of introgressed japonica components (Figure 4). This finding suggests that the Fj values calculated based on the "InDel molecular index" (Table S1) could be used to estimate the level of japonica-specific allelic introgression for further analyses, particularly the relationships of crop-to-weed introgression with genetic differentiation and genetic diversity in JS weedy rice based on the Fj values.

Genetic Differentiation in Weedy Rice Associated with Crop-to-Weed Introgression
To investigate the patterns of genetic differentiation in JS weedy rice samples, we conducted the principal coordinate analysis (PCoA) based on the InDel molecular fingerprints, using typical indica and japonica rice varieties and weedy rice samples from GD and NEC as references. The PCoA results demonstrated evident genetic differentiation of JS weedy rice samples into indica and japonica types ( Figure 5). Obviously, most weedy rice samples from JS were scattered and were closely associated with the typical indica rice

Genetic Differentiation in Weedy Rice Associated with Crop-to-Weed Introgression
To investigate the patterns of genetic differentiation in JS weedy rice samples, we conducted the principal coordinate analysis (PCoA) based on the InDel molecular fingerprints, using typical indica and japonica rice varieties and weedy rice samples from GD and NEC as references. The PCoA results demonstrated evident genetic differentiation of JS weedy rice samples into indica and japonica types ( Figure 5). Obviously, most weedy rice samples from JS were scattered and were closely associated with the typical indica rice varieties and the weedy rice samples from GD, at the negative loads of the first principal coordinate (left in Figure 5). Therefore, these weedy rice samples were likely the indica type, which was supported by their low F j values (<0.25, Table 1). A small proportion of weedy rice samples from JS was scattered among the typical japonica rice varieties and the weedy rice samples from NEC (references), at the positive loads of the first principal coordinate (right in Figure 5). Therefore, these weedy rice samples were most likely the japonica type, which was also supported by their high F j values (>0.75, Table 1). Noticeably, some of the JS weedy rice samples were scattered between the typical indica and japonica types along the first principal coordinate (middle in Figure 5), which were determined as the indica-japonica intermediate types with the F j value between 0.25 and 0.75 (Table 1). weedy rice samples from NEC (references), at the positive loads of the first principal coordinate (right in Figure 5). Therefore, these weedy rice samples were most likely the japonica type, which was also supported by their high Fj values (>0.75, Table 1). Noticeably, some of the JS weedy rice samples were scattered between the typical indica and japonica types along the first principal coordinate (middle in Figure 5), which were determined as the indica-japonica intermediate types with the Fj value between 0.25 and 0.75 (Table 1). In general, the PCoA results clearly indicate the genetic differentiation of JS weedy rice samples that are scattered between the typical reference indica and japonica rice varieties. This finding was supported by the gradually increased japonica-specific allelic frequency (Fj) in the JS weedy rice samples that should originally be the indica type.
To estimate the correlation between the extent/level of genetic differentiation and crop-to-weed introgression, we calculated the pairwise genetic differentiation (Fst) and differences in allelic frequency (Fj-d), based on the data matrices of InDel (Figures 6a and  7a) and SSR (Figures 6b and 7b) molecular fingerprints. The correlation analysis was conducted based on the 30 ideally sampled weedy rice populations (ISWPs) with their respective low, middle, and high levels of introgression (Figure 6), and eight natural weedy rice populations (NSWPs) (Figure 7) for both InDel and SSR molecular fingerprints. Results based on the Pearson's correlation analysis showed a significantly positive correlation between genetic differentiation as measured by the pairwise Fst values and crop-to-weed introgression as estimated by the pairwise Fj-d values of ISWPs (R 2 = 0.94-0.96, p < 0.001) and NSWPs (R 2 = 0.35-0.73, p < 0.01). These results generated from the ISWPs and NSWPs of JS weedy rice suggest that crop allelic introgression would cause considerable genetic differentiation of its conspecific weed. In general, the PCoA results clearly indicate the genetic differentiation of JS weedy rice samples that are scattered between the typical reference indica and japonica rice varieties. This finding was supported by the gradually increased japonica-specific allelic frequency (F j ) in the JS weedy rice samples that should originally be the indica type.
To estimate the correlation between the extent/level of genetic differentiation and crop-to-weed introgression, we calculated the pairwise genetic differentiation (F st ) and differences in allelic frequency (F j-d ), based on the data matrices of InDel (Figures 6a and 7a) and SSR (Figures 6b and 7b) molecular fingerprints. The correlation analysis was conducted based on the 30 ideally sampled weedy rice populations (ISWPs) with their respective low, middle, and high levels of introgression (Figure 6), and eight natural weedy rice populations (NSWPs) (Figure 7) for both InDel and SSR molecular fingerprints. Results based on the Pearson's correlation analysis showed a significantly positive correlation between genetic differentiation as measured by the pairwise F st values and crop-to-weed introgression as estimated by the pairwise F j-d values of ISWPs (R 2 = 0.94-0.96, p < 0.001) and NSWPs (R 2 = 0.35-0.73, p < 0.01). These results generated from the ISWPs and NSWPs of JS weedy rice suggest that crop allelic introgression would cause considerable genetic differentiation of its conspecific weed.

Genetic Diversity of Weedy Rice Associated with Crop-to-Weed Introgression
To estimate the correlation/relationship between the level of genetic diversity and crop-to-weed introgression, we calculated Nei's expected heterozygosity (He) and Shannon's information index (I) to represent genetic diversity, based on the data matrices of InDel and SSR molecular fingerprints. In addition, we used the frequency of japonica-specific alleles (Fj) to represent the level of crop-to-weed introgression. The correlation analysis was conducted based on the 30 ISWPs with their respective low, middle, and high levels of introgression ( Figure 8) for both InDel and SSR molecular fingerprints. Results based on the quadratic equation fitting regression analysis indicated a high degree of fitting (R 2 = 0.98 for He and 0.99 for I) for the InDel molecular fingerprints (Figure 8a,c). Similarly, the quadratic equation fitting regression analysis indicated a relatively high degree of fitting (R 2 = 0.81 for He and 0.78 for I) for the SSR molecular fingerprints (Figure 8b,d).

Genetic Diversity of Weedy Rice Associated with Crop-to-Weed Introgression
To estimate the correlation/relationship between the level of genetic diversity and cropto-weed introgression, we calculated Nei's expected heterozygosity (H e ) and Shannon's information index (I) to represent genetic diversity, based on the data matrices of InDel and SSR molecular fingerprints. In addition, we used the frequency of japonica-specific alleles (F j ) to represent the level of crop-to-weed introgression. The correlation analysis was conducted based on the 30 ISWPs with their respective low, middle, and high levels of introgression ( Figure 8) for both InDel and SSR molecular fingerprints. Results based on the quadratic equation fitting regression analysis indicated a high degree of fitting (R 2 = 0.98 for H e and 0.99 for I) for the InDel molecular fingerprints (Figure 8a,c). Similarly, the quadratic equation fitting regression analysis indicated a relatively high degree of fitting (R 2 = 0.81 for H e and 0.78 for I) for the SSR molecular fingerprints (Figure 8b,d).
Biology 2023, 12, x FOR PEER REVIEW 12 of 19 Figure 8. Results of regression between genetic diversity represented by Nei's expected heterozygosity (He) and Shannon's information index (I) [59,60] and the frequency of japonica-specific alleles (Fj) [43], based on the insertion/deletion (InDel, (a) and (c)) and simple sequence repeat (SSR, (b) and (d)) data matrices of the ideally sampled weedy rice populations (empty dots). Black curves are regression lines.
In addition, the correlation between the level of genetic diversity (He and I) and cropto-weed introgression (Fj) was also analyzed based on the eight NSWPs (Figure 9). Results Figure 8. Results of regression between genetic diversity represented by Nei's expected heterozygosity (H e ) and Shannon's information index (I) [59,60] and the frequency of japonica-specific alleles (F j ) [43], based on the insertion/deletion (InDel, (a,c)) and simple sequence repeat (SSR, (b,d)) data matrices of the ideally sampled weedy rice populations (empty dots). Black curves are regression lines.
In addition, the correlation between the level of genetic diversity (H e and I) and crop-to-weed introgression (F j ) was also analyzed based on the eight NSWPs (Figure 9). Results showed that the level of genetic diversity (H e and I) significantly increased with the increases in the F j values for the InDel molecular fingerprints (Figure 9a,c), when F j varied between 0.05 and 0.34. However, no significant correlations were observed between the level of genetic diversity (H e and I) and the F j values for the SSR molecular fingerprints (Figure 9b,d). The relationship between the level of genetic diversity and crop-to-weed introgression (represented by the F j values) generated based on these results from NSWPs generally agreed with those revealed from ISWPs, particularly for InDel molecular fingerprints.

The Change in Rice Varieties Greatly Influences Indica-Japonica Characteristics of Weedy Rice through Crop-to-Weedy Introgression
Our results in this study based on the historical archives clearly demonstrated t pattern of traditional rice cultivation in Jiangsu Province (JS), where indica varieties we grown essentially until the early 1950s. Different japonica rice varieties were gradually troduced into this province at the beginning of the 1960s because of their favorable co mercial quality (taste) and high yield [35][36][37]. After three decades by the 1990s, the ind rice varieties were almost completely replaced by japonica rice varieties in different r cultivation areas in this province. As a consequence, weedy rice with japonica character tics was reported to appear frequently in the rice fields of this province [23,33,42]. App ently, the changing pattern of rice cultivation from indica to japonica varieties has cons erable influences on the genetic compositions of weedy rice as indicated by its indica a japonica characteristics. Such influences are hypothetically through gene flow or allelic trogression from japonica rice varieties to weedy rice individuals.
In fact, our results showed that most examined JS weedy rice samples (individua were the indica type (~91%) and the rest of the weedy rice samples were the intermedia Figure 9. Correlations as represented by dashed lines (regression) between the genetic diversity parameters (H e , I) [59,60] and the F j values [43] of the eight naturally sampled weedy rice populations (NSWPs) from Jiangsu Province. The F j values and the genetic diversity parameters were calculated based on the data matrixes from the InDel (a,c) and SSR (b,d) molecular fingerprints, respectively. Each column represents an NSWP (n = 31), and the error bars indicate standard errors.

The Change in Rice Varieties Greatly Influences Indica-Japonica Characteristics of Weedy Rice through Crop-to-Weedy Introgression
Our results in this study based on the historical archives clearly demonstrated the pattern of traditional rice cultivation in Jiangsu Province (JS), where indica varieties were grown essentially until the early 1950s. Different japonica rice varieties were gradually introduced into this province at the beginning of the 1960s because of their favorable commercial quality (taste) and high yield [35][36][37]. After three decades by the 1990s, the indica rice varieties were almost completely replaced by japonica rice varieties in different rice cultivation areas in this province. As a consequence, weedy rice with japonica characteristics was reported to appear frequently in the rice fields of this province [23,33,42]. Apparently, the changing pattern of rice cultivation from indica to japonica varieties has considerable influences on the genetic compositions of weedy rice as indicated by its indica and japonica characteristics. Such influences are hypothetically through gene flow or allelic introgression from japonica rice varieties to weedy rice individuals.
In fact, our results showed that most examined JS weedy rice samples (individuals) were the indica type (~91%) and the rest of the weedy rice samples were the intermediate and japonica types, based on the InDel molecular index [43] or the average frequency of the japonica-specific alleles (F j ). These results were obtained based on a relatively large number of weedy rice samples (1116 individuals) collected from 36 populations across the JS rice cultivation areas. This finding indicates the change in characteristics of JS weedy rice gradually from the original indica type to the japonica and indica-japonica intermediate types, associated with the rapid changes in rice cultivation patterns from indica to japonica varieties in this province. In other words, the rapid alteration of rice cultivation from indica to japonica varieties promoted the divergence of JS weedy rice characteristics, essentially through crop-to-weed introgression: although, other factors, such as direct-seeding and mechanic harvesting, cannot be completely excluded. The changes in the indica-japonica characteristics of JS weedy rice found in this study are similar to those revealed either by phenotypical characterization [42,61] or molecular fingerprinting [23,34], in which weedy rice was composed of the indica, japonica, and intermediate types, although predominated by the indica type. All results clearly indicate a wide range of variation regarding the indica-japonica characteristics of JS weedy rice, owing to the rice cultivation change from indica to japonica varieties.
In this study, we also found that the reference weedy rice populations from NEC were essentially the japonica type that was associated strongly with the typical japonica rice varieties, whereas those from GD were essentially the indica type that was associated intimately with the typical indica rice varieties. Our finding is consistent with the previous reports in which weedy rice is genetically closely associated with its cultivated counterparts co-occurring in the same regions [21,23,27,31]. Obviously, the JS weedy rice samples with different genetic backgrounds identified in this study should be the result of successive gene flow or introgression with different types of indica and japonica rice varieties cultivated during a historical period of time in this province. Successive crop-to-weed gene flow or introgression, coupled with the independent assortment and genetic recombination in the self-pollination process, promoted the admixture of indica and japonica genotypes in JS weedy rice.
Results from the STRUCTURE analysis based on the InDel molecular fingerprints in this study clearly demonstrated the change in genetic compositions in JS weedy rice from the original indica type to the current japonica types with evident admixture genetic components, as indicated by the different bar-plots with three K values ( Figure 3). The presence of the indica-japonica admixture types of weedy rice in the JS rice cultivation areas confirmed crop-to-weed allelic introgression, because of the accumulation of japonica alleles in the admixture types of JS weedy rice samples. The presence of indica-japonica admixture types in the JS weedy rice samples can also exclude the possible contamination of these samples as japonica weedy rice seeds. This is because the mixed japonica weedy rice samples, if any, in the certified commercial japonica cultivar seeds, should be present as the pure japonica genotype in the STRUCTURE analysis. Somehow, there is a possibility of the contamination of a few weedy rice seeds with the newly introduced japonica rice varieties in the actual rice production. Given that the opportunities for inter-crosses between the distantly scattered weedy rice plants are very low, the gradual accumulation of japonica alleles in weedy rice is more likely through crop-to-weed introgression [62][63][64][65][66][67][68] from japonica rice varieties, rather than by spontaneous mutations or through seed contamination. In addition, the gradual change in JS weedy rice genetic components from the original indica type to the admixture and japonica types matches the pattern of JS rice variety change in cultivation for the past seven decades (Figure 2). The relatively low proportion of JS weedy rice samples with japonica-specific alleles from their cooccurring cultivars can be explained by the inbreeding feature of weedy rice with a relatively low outcrossing rate (~1%) [62][63][64][65][66][67][68]. Thus, we propose that crop-to-weed introgression has played an important role not only in changing the indica-japonica characteristics and genetic components of JS weedy rice, but also in shaping the evolution of JS weedy rice as an important driving force. These observations agree with the previous reports concerning weedy rice genetic diversity and evolution [17][18][19][20][21][22].

Crop-to-Weed Introgression Impacts Genetic Differentiation and Genetic Diversity in Weedy Rice through Accumilated Crop-Specific Alleles
Our results clearly indicate that the level of gene flow or crop-to-weed introgression can be measured by the frequency of the japonica-specific alleles (F j ) of the weedy rice samples with considerably high confidence (Figure 4). Results based on the PCoA analysis of the InDel molecular fingerprints also demonstrated substantial genetic differentiation of JS weedy rice, which was somehow associated with crop-to-weed introgression caused by the change in rice cultivation for japonica varieties. Given that the F j value of each weedy rice sample can be accurately calculated based on the InDel molecular fingerprinting [43], we analyzed the impact of crop-to-weed introgression on genetic differentiation and genetic diversity of weedy rice. The analysis of such impact can be realized through calculating the correlation between the F j values and the level of genetic differentiation (F st ), as well as genetic diversity (H e and I), using the ideally sampled weedy rice populations (ISWPs) established according to their levels (low, middle, and high) of introgression determined by the F j values.
Further analyses in this study indicated that the level of japonica-specific allelic (cropto-weed) introgression was highly significantly correlated (R 2 = 0.94, p < 0.001) with the level of F j values of the examined weedy rice samples. Therefore, the F j values can represent japonica-specific allelic introgression and be directly utilized to determine the relationships between the level of crop-to-weed introgression and the genetic differentiation of weedy rice. Results obtained based on ISWPs with low, middle, and high levels of introgression, using both the InDel and SSR molecular fingerprints, clearly demonstrated the correlation patterns, in which crop-to-weed introgression substantially prompted the genetic differentiation of JS weedy rice. Interestingly, similar correlation patterns were obtained based on the randomly selected naturally sampled weedy rice populations (NSWPs) with low to middle levels of introgression in JS, although with a much lower level of correlation for the results obtained using the SSR molecular fingerprints. Therefore, we can conclude based on all results from this study that crop-to-weed introgression can significantly promote the genetic differentiation of weedy rice populations with the original indica genotype through accumulating japonica-specific alleles from its co-existing japonica rice varieties. In other words, the gradual introgression of different crop-specific alleles into weedy rice populations can considerably cause their within-and between-population genetic differentiation.
Our results based on the InDel molecular fingerprints also suggest that the level of F j values was significantly associated with the two independent genetic diversity parameters (Nei's expected heterozygosity, H e , and Shannon's information index, I) in both ISWPs and NSWPs (Figures 8 and 9). Noticeably, the polymorphic SSR molecular markers did not show a significant correlation between genetic diversity and introgression in NSWPs, in addition to a comparably lower level of correlation than the dimorphic (indica-japonica) InDel molecular markers. The differences in genetic diversity revealed by SSR and InDel molecular markers can easily be explained by the reasons that the formation of genetic diversity in weedy rice is not only determined by crop-to-weed introgression involving indica-japonica alleles or characteristics, but also by other types of alleles that are not associated with the indica and japonica characteristics. Therefore, we propose that, based on this case study, the F j values that can represent japonica-specific allelic introgression can also be used to determine the relationships between the level of crop-to-weed introgression and the dynamics of genetic diversity in weedy rice populations. This conclusion is supported by previous studies, in which an increased level of crop-to-weed introgression can promote rapid changes in genetic diversity patterns in weedy populations [19,20,23,27].

Human Activities Can Accelerate the Evolution of Conspecific Weeds in Agroecosystems
It is well-known that weedy rice infests worldwide rice fields, causing considerable losses in the grain yield and quality of cultivated rice [25][26][27]. As a conspecific weed of cultivated rice, weedy rice evolved rapidly in rice ecosystems to adapt to the weed control and the environmental changes associated with human activities around the globe [18,[25][26][27].
Weedy rice has become a great weed problem for rice cultivation around the world, including in the rice-planting regions in China (e.g., Jiangsu Province), which threatens sustainable rice production [33,42,69]. In some regions, a rapid change in the rice ecosystems has taken place, such as the shift from rice transplanting to direct seeding, the application of farming machinery, and the quick change in rice varieties [35][36][37]69]. These human activities in the rice ecosystems could have imposed a strong impact on the evolutionary processes of weeds, including weedy rice, to adapt to the changing environment. This case study that we completed in Jiangsu Province with documented changes in rice cultivation from indica to japonica varieties for the past few decades reveals the human-influenced evolution of weedy rice in agroecosystems.
Our results indicated the presence of the japonica-specific alleles in JS weedy rice, most likely through crop-to-weed allelic introgression based on the InDel molecular fingerprints. Such introgression is closely associated with the rapid replacement of indica rice varieties by japonica rice varieties in JS. This finding evidently indicates that human-influenced cultivation changes in rice varieties (from indica to japonica) alone have already altered the genetic components of the co-occurring weedy rice populations. Our results from the analysis of InDel and SSR molecular fingerprints based on the ISWPs and NSWPs further demonstrated a positive correlation of crop-to-weed introgression, as measured by the F j values, with the genetic differentiation of weedy rice. In addition, these results also confirmed the close association between the levels of crop-to-weed introgression and genetic diversity both in ISWPs and NSWPs, using the two sets of molecular fingerprints.
Altogether, these findings demonstrated that human activities or disturbances can significantly influence the genetic differentiation and genetic diversity of weedy rice-a conspecific weed-only through consecutive allelic introgression from its cooccurring cultivated counterparts that have constantly been improved by humans at different periods in time. This conclusion is supported by the large number of japonica-specific alleles detected in JS weedy rice, which is associated with the rapid change in rice cultivation from indica to japonica varieties. Genetic differentiation and genetic diversity are two important elements that are closely associated with the evolution of plant species. Therefore, we consider that human activities or disturbances can promote the rapid adaptive evolution of conspecific weedy rice in rice ecosystems through the change in these elements, which makes the control and management of weedy rice very difficult.
Such human-influenced rapid evolution of agricultural weeds as revealed in this case study may also be frequently found in many other weedy plant species in agroecosystems [8,15,16,70,71]. Previous studies reported that crop-weed introgression promoted an increase in genetic diversity in cooccurring weed populations [23,28,71,72], due to the frequent changes in newly developed crop varieties around the world [23,24,73]. Very often, these new varieties contained many new alleles/genes, with even transgenes having great evolutionary potential [17,24,73,74]. The results presented in this study provide a convincing case to explain how a conspecific weed can evolve rapidly by accumulated crop alleles from diverse crop varieties through crop-to-weed introgression to promote its genetic differentiation and diversity. Such an impact on the adaptive evolution of conspecific weeds imposes a great challenge for the control and management of these weeds.

Conclusions
In this study, we detected many japonica-specific alleles in JS weedy rice that should originally be the indica type with the indica genetic background, based on the insertion/deletion (InDel) molecular fingerprints. The presence of the japonica-specific alleles in the indica type of weedy rice is most likely the result of gene flow or introgression of alleles from japonica rice varieties; although, in the practical rice production, a very low frequency of japonica weedy rice contamination might also happen. Such gradual introgression of japonica alleles is closely associated with the change in rice cultivation from indica to japonica varieties in the past few decades. Our results further indicate that crop-to-weed allelic introgression has considerably changed the genetic components of the cooccurring JS-weedy rice populations. Further analyses based on InDel and SSR molecular fingerprinting indicate a significant positive correlation between the levels of crop-to-weed introgression and genetic differentiation in JS weedy rice. Similarly, increased crop-to-weed introgression promoted a change in genetic diversity in weedy rice with a parabola correlation pattern. Altogether, the above findings demonstrate that human activities, such as the change in cultivated rice varieties, can impose a considerable impact on the evolution of its conspecific weed by promoting genetic differentiation and diversity in rice ecosystems. A similar pattern of crop-to-weed introgression promoting genetic differentiation and genetic diversity is likely found in other crops and their conspecific weeds. Therefore, we conclude that, based on this case study, human activities or disturbances may accelerate the adaptive evolution of conspecific weeds through crop-to-weed introgression in agroecosystems, which may impose a great challenge for the control and management of these weeds.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/biology12050744/s1, Table S1, The localities, GPS readings, indica-japonica characteristics (F j ) of weedy rice and cultivated rice samples used in this study; Table S2, DNA sequences of InDel and SSR primer pairs; Table S3, Average planting areas of indica and japonica rice varieties for every 10 years (khm 2 ).